Process for producing coenzymes q10

ABSTRACT

The invention aims at providing a process for producing coenzyme Q 10  efficiently in microorganisms by utilizing a coenzyme Q 10  side chain synthesis gene derived from a fungal species belonging to the genus Aspergillus and genus Leucosporidium.  
     The present invention relates to a DNA having a DNA sequence described under SEQ ID NO:1 and 2 or derived from the above sequence by deletion, addition, insertion and/or substitution of one or several bases and encoding a protein having decaprenyl diphosphate synthase activity.

TECHNICAL FIELD

[0001] The present invention relates to a process for producing coenzyme Q₁₀, which is in use as a drug or the like. More particularly, it relates to a process for causing formation of coenzyme Q₁₀ by introducing a gene coding for a coenzyme Q₁₀ side chain synthase, which serves as a key enzyme in the biosynthesis of coenzyme Q₁₀, namely a decaprenyl diphosphate synthase into a microorganism.

BACKGROUND ART

[0002] An industrial process for producing coenzyme Q₁₀, which is conventional in the art, comprises, for example, isolating coenzymes of plant origin, for example of tobacco origin, and adjusting the side chain length thereof by a synthetic method.

[0003] It is known that coenzyme Q₁₀ is produced in a wide variety of organisms, from microorganisms, such as bacteria and yeasts, to higher animals and plants. Thus, the process comprising cultivating a microorganism and extracting this substance from cells thereof can be regarded as one of the most efficient process for producing coenzyme Q₁₀ and has actually been employed in commercial production thereof. However, the productivity of such processes can hardly be said to be good, since the yield is low and the procedure is complicated, for instance.

[0004] Attempts have also been made to increase the production of coenzyme Q₁₀ by isolating genes involved in the biosynthesis of coenzyme Q₁₀ and amplifying the genes utilizing the recombinant DNA technology. Coenzyme Q₁₀ is formed in vivo in a multistage process comprising complicated reactions in which a number of enzymes are involved. The route of biosynthesis thereof in prokaryotes partially differs from that in eukaryotes. Basically, however, each route comprises three fundamental steps, namely the step of synthesis of decaprenyl diphosphate, which is the source of the prenyl side chain of coenzyme Q₁₀, the step of synthesis of para-hydroxybenzoic acid, which is the source of the quinone ring, and the step of completion of coenzyme Q₁₀ through coupling of these two compounds and successive substituent conversions. Among these reactions, the reaction which determines the side chain length of coenzyme Q₁₀, namely the decaprenyl diphosphate synthase-involving reaction, which is said to be a rate-determining one in the whole biosynthetic reaction route, is considered to be the most important one.

[0005] Therefore, for efficient production of coenzyme Q₁₀, it is considered effective to isolate a decaprenyl diphosphate synthase gene, which is the key gene in the biosynthesis of coenzyme Q₁₀, and utilize the same for the purpose of increasing production. As for the gene source, fungi, in which coenzyme Q₁₀ is produced in relatively large amounts, are leading candidates.

[0006] So far, decaprenyl diphosphate synthase genes have been isolated from several microorganisms, such as Schizosaccharomyces pombe (JP-A-09-173076) and Gluconobacter suboxydans (JP-A-10-57072). However, the productivity of coenzyme Q₁₀ in these microorganisms cannot be said to be satisfactory, and the cultivation of these microorganisms and the separation/purification of coenzyme Q₁₀ therefrom have not been efficient. It has thus been desired that a microorganism-derived gene for that enzyme, which enables high level production of coenzyme Q₁₀, be isolated.

SUMMARY OF THE INVENTION

[0007] It is an object of the present invention to produce coenzyme Q₁₀ efficiently in microorganisms by isolating a coenzyme Q₁₀ side chain synthesis gene from a fungal species belonging to the genus Leucosporidium and genus Aspergillus, and utilizing the same to thereby solve the above-mentioned productivity problem.

[0008] For attaining the above object, in the present invention, firstly, investigations were made in an attempt to isolate a key gene involved in the biosynthesis of coenzyme Q₁₀, a decaprenyl diphosphate synthase gene from fungi belonging to the genus Leucosporidium and genus Aspergillus and succeeded in isolating such gene. This success has now led to completion of the present invention.

[0009] Thus, the present invention provides

[0010] a DNA of the following (a), (b) or (c):

[0011] (a) a DNA whose base or sequence is as described under SEQ ID NO:1;

[0012] (b) a DNA having a base sequence derived from the base sequence shown under SEQ ID NO:1 by deletion, addition, insertion and/or substitution of one or several bases and

[0013] encoding a protein having decaprenyl diphosphate synthase activity;

[0014] (c) a DNA capable of hybridizing with a DNA comprising the base sequence shown under SEQ ID NO:1 under stringent conditions and

[0015] encoding a protein having decaprenyl diphosphate synthase activity.

[0016] The invention further provides

[0017] a DNA of the following (d), (e) or (f):

[0018] (d) a DNA whose base sequence is as described under SEQ ID NO:2;

[0019] (e) a DNA having a DNA sequence derived from the base sequence shown under SEQ ID NO:2 by deletion, addition, insertion and/or substitution of one or several bases and

[0020] encoding a protein having decaprenyl diphosphate synthase activity;

[0021] (f) a DNA capable of hybridizing with a DNA comprising the base sequence shown under SEQ ID NO:2 under stringent conditions and

[0022] encoding a protein having decaprenyl diphosphate synthase activity.

[0023] The invention further provides

[0024] a protein of the following (g) or (h):

[0025] (g) a protein whose amino acid sequence is as described under SEQ ID NO:3;

[0026] (h) a protein having an amino acid sequence derived from the amino acid sequence shown under SEQ ID NO:3 by deletion, addition, insertion and/or substitution of one or several amino acid residues and having decaprenyl diphosphate synthase activity.

[0027] The invention further provides

[0028] a protein of the following (i) or (j):

[0029] (i) a protein whose amino acid sequence is as described under SEQ ID NO:4;

[0030] (j) a protein having an amino acid sequence derived from the amino acid sequence shown under SEQ ID NO:4 by deletion, addition, insertion and/or substitution of one or several amino acid residues and having decaprenyl diphosphate synthase activity.

[0031] The invention further provides

[0032] DNAs respectively encoding the above proteins (g) to (j).

[0033] The invention further provides

[0034] expression vectors with the above DNAs inserted into vectors.

[0035] The invention further provides

[0036] transformants resulting from transformation of host microorganisms with the respective DNAs mentioned above or with the expression vectors mentioned above.

[0037] The invention still further provides

[0038] a process for producing coenzyme Q₁₀,

[0039] which comprises cultivating any of the above-mentioned transformants in a medium and recovering coenzyme Q₁₀ thus formed and accumulated in the medium.

DETAILED DISCLOSURE OF THE INVENTION

[0040] The present inventors made investigations to isolate a gene encoding the enzyme in question from fungi belonging to the genus Leucosporidium and genus Aspergillus, in which coenzyme Q₁₀ is produced in relatively large amounts, and, as a result, succeeded in obtaining a fragment of the gene by the PCR method.

[0041] The sequence of a known gene encoding a decaprenyl diphosphate synthase was compared with that of a known gene encoding a polyprenyl diphosphate synthase which is an analogue of the enzyme in question and a long prenyl chain synthase of different chain length coenzyme Q, and various PCR primers were synthesized for regions showing high homology therebetween. PCR conditions were studied for various combinations of these primers and, as a result, it was revealed, by gene base sequence analysis, that when 40 PCR cycles, each comprising 94° C., 1 minute→43° C., 2 minutes→72° C., 2 minutes, are carried out after 3 minutes of heat treatment at 94° C., using the primers DPS-1 (SEQ ID NO:5 in the sequence listing) and DPS-1 1AS (SEQ ID NO:6 in the sequence listing), a fragment, about 220 bp in size, of the enzyme gene in question is amplified from the chromosomal gene of Leucosporidium scotti IFO 1212, which is a fungal species belonging to the genus Leucosporidium and Aspergillus clavatus JCM 1718, which is a fungal species belonging to the genus Aspergillus.

[0042] For obtaining the enzyme gene in its full length, mRNA is first prepared from a fungus body of Leucosporidium scotti IFO 1212 and Aspergillus clavatus JCM 1718 in order to respectively produce 5′ terminal side sequence by 5′RACE method using a primer prepared based on the inner sequence obtained before and produce 3′ terminal side sequence by conducting RT-PCR using oligo dT primer relative to a poly A sequence, which is specific for the mRNA.

[0043] The base sequence of the sequence obtained was determined, whereupon it was revealed that it has the sequence shown under SEQ ID NO:1 and NO:2 in the sequence listing. In the amino acid sequence predicted from this base sequence, there was found a sequence characteristic of decaprenyl diphosphate synthase.

[0044] The DNA of the present invention may be a DNA whose base sequence is as described under SEQ ID NO:1 or SEQ ID NO:2, or a DNA having a base sequence derived from the base sequence shown under SEQ ID NO:1 or SEQ ID NO:2 by deletion, addition, insertion and/or substitution of one or several bases and encoding a protein having decaprenyl diphosphate synthase activity, or a DNA capable of hybridizing with a DNA comprising the base sequence shown under SEQ ID NO:1 or SEQ ID NO:2 under stringent conditions and encoding a protein having decaprenyl diphosphate synthase activity. A number of amino acids each may be encoded by one or more codons (genetic code degeneracy), so that a number of DNAs other than the DNA having the base sequence shown under SEQ ID NO:1 or SEQ ID NO:2 can encode the protein having the amino acid sequence shown under SEQ ID NO:3 or SEQ ID NO:4. Therefore, the DNA of the invention includes such DNAs encoding the protein having the amino acid sequence shown under SEQ ID NO:3 or SEQ ID NO:4 as well.

[0045] The expression “base sequence derived by deletion, addition, insertion and/or substitution of one or several bases” as used herein means a base sequence resulting from deletion, addition, insertion and/or substitution of such a number of bases as can be deleted, added, inserted and/or substituted according to the methods well known to those skilled in the art, for example those described in Supplemental issue, Tanpakushitsu, Kakusan, Koso (Protein, Nucleic Acid and Enzyme), PCR Method for Gene Amplification, TAKKAJ, 35 (17), 2951-3178 (1990) or Henry A. Erlich (ed.), translated into Japanese under the supervision of Ikunoshin Kato: PCR Technology (1990).

[0046] The expression “DNA capable of hybridizing with a DNA comprising the base sequence shown under SEQ ID NO:1 (or SEQ ID NO:2) under stringent conditions” means a DNA obtainable by utilizing the technique of colony hybridization, plaque hybridization or southern hybridization, among others, using a DNA comprising the base sequence shown under SEQ ID NO:1 (or SEQ ID NO:2) as a probe. Those skilled in the art would be able to readily obtain the desired DNA by carrying out such hybridization according to the method described in Molecular Cloning, 2nd edition (Cold Spring Harbor Laboratry Press, 1989).

[0047] The expression “protein having decaprenyl diphosphate synthase activity” means a protein capable of synthesizing decaprenyl diphosphate in a yield of not less than 10%, preferably not less than 40%, more preferably not less than 60%, still more preferably not less than 80%, as compared with the case where a protein having the amino acid sequence shown under SEQ ID NO:3 or SEQ ID NO:4. Such capacity can be measured by reacting FPP (farnesyl diphosphate) with ¹⁴C-IPP (radiolabeled isopentenyl diphosphate) in the presence of the enzyme in question, hydrolyzing the resulting ¹⁴C-DPP (decaprenyl diphosphate) with phosphatase and, after separation by TLC, determining the incorporation in each spot for each chain length (Okada et al., Eur. J. Biochem., 255, 52-59).

[0048] The protein of the invention may be a protein whose amino acid sequence is as described under SEQ ID NO:3 or SEQ ID NO:4, or a protein having an amino acid sequence derived from the amino acid sequence shown under SEQ ID NO:3 or SEQ ID NO:4 by deletion, addition, insertion and/or substitution of one or several amino acid residues and having decaprenyl diphosphate synthase activity.

[0049] “Such an amino acid sequence derived by deletion, addition, insertion and/or substitution of one or several amino acid residues” can be obtained by deleting, adding, inserting and/or substituting an amino acid residue or residues by site-specific mutagenesis or any other methods well known in the art. Such methods are specifically described, for example, in Nucleic Acid Res., 10, 6487 (1982) and Methods in Enzymology, 100, 448 (1983).

[0050] For causing expression of the decaprenyl diphosphate synthase gene, it is necessary to connect that gene to a site downstream of an appropriate promoter. It is possible to construct an expression vector, for example, by excising a DNA fragment containing the gene using a restriction enzyme or amplifying an enzyme-encoding gene portion alone by PCR and then inserting the fragment or amplification product into a promoter-containing vector.

[0051] In the practice of the invention, the vector in which a DNA encoding a protein having decaprenyl diphosphate synthase activity is to be inserted to give an expression vector is not particularly restricted but may be one derived from an Escherichia coli-derived plasmid with an appropriate promoter inserted therein. The Escherichia coli-derived plasmid includes, among others, pBR322, pBR325, pUC19, pUC18 and pUC119, and the promoter includes, among others, the T7 promoter, trp promoter, tac promoter, lac promoter, and λPL promoter.

[0052] In the practice of the invention, pGEX-2T, pGEX-3T, pGEX-3X (the three being products of Pharmacia), pBluescriptII, pUC19, pUC18 (product of Toyobo Co., Ltd.), pMALC2, pET-3T, pUCNT (described in Publication WO 94/03613) and the like may also be used as vectors for expression. Among these, pUCNT and pUC18 are judiciously used. In specific examples, a decaprenyl diphosphate synthase gene expression vector, pNTL1, can be constructed by inserting a gene containing the DNA sequence shown under SEQ ID NO:1 into the vector for expression pUCNT, and an expression vector of decaprenyl diphosphate synthase gene, pUCA1, can be constructed when a gene containing the DNA sequence shown under SEQ ID NO:2 is inserted into the vector for expression pUC18.

[0053] And, by introducing the above enzyme gene expression vector into an appropriate microorganism, it becomes possible to utilize the microorganism for the production of coenzyme Q₁₀. The host microorganism is not particularly restricted but Escherichia coli is judiciously used. The Escherichia coli strain is not particularly restricted but includes XL1-Blue, BL-21, JM109, NM522, DH5α, HB101, and DH5, among others. Among these, Escherichia coli DH5α and JM109 are judiciously used. For example, when the decaprenyl diphosphate synthase gene expression vector pNTL1 is introduced into Escherichia coli, this can be transformed so that coenzyme Q₁₀, which Escherichia coli originally does not produce, can be produced. The Escherichia coli strain E. coli DH5α (pNTL1) has been deposited with the National Institute of Advanced Industrial Science and Technology International Patent Organism Depositary (zip code: 305-8566 Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan) under the designation FERM BP-7353. Further, when the decaprenyl diphosphate synthase gene expression vector pUCA1 is introduced into Escherichia coli, this can be transformed so that coenzyme Q₁₀, which Escherichia coli originally does not produce, can be produced. The Escherichia coli strain E. coli JM109 (pUCA1) has been deposited with the National Institute of Advanced Industrial Science and Technology International Patent Organism Depositary (zip code: 305-8566 Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan) under the designation FERM BP-7352.

[0054] Coenzyme Q₁₀ can be produced by cultivating the transformant obtained in the invention in the conventional manner and recovering coenzyme Q₁₀ from the cultivation product. In cases where the host microorganism is Escherichia coli, LB medium, or M9 medium containing glucose and casamino acids can be used as the medium. For better promoter functioning, such an agent as isopropylthiogalactoside or indolyl-3-acrylic acid, for instance, may be added to the medium. The cultivation is carried out, for example, at 37° C. for 17 to 24 hours, if necessary with aeration and/or agitation.

[0055] In the practice of the invention, the product coenzyme Q₁₀ obtained may be purified or used in the form of a crude product according to the selection duly made depending on the intended use thereof. Coenzyme Q₁₀ can be isolated from the cultivation product by an appropriate combination of per se known methods of separation and/or purification. The per se known methods of separation and/or purification include salting out, solvent precipitation and other methods utilizing the difference in solubility, dialysis, ultrafiltration, gel filtration, (SDS-)polyacrylamide gel electrophoresis and other methods mainly utilizing the difference in molecular weight, ion exchange chromatography and other methods utilizing the difference in charge, affinity chromatography and other methods utilizing specific affinity, reversed phase high-performance liquid chromatography and other methods utilizing the difference in hydrophobicity, isoelectric focusing and other methods utilizing the difference in isoelectric point, among others.

[0056] The field of utilization of coenzyme Q₁₀ obtained in the present invention is not particularly restricted but it may be judiciously used as a drug, among others.

BRIEF DESCRIPTION OF THE DRAWINGS

[0057]FIG. 1 is a restriction enzyme map of the expression vector pNTL1.

[0058]FIG. 2 is a restriction enzyme map of the expression vector pUCA1.

[0059]FIG. 3 shows HPLC analysis charts showing production of coenzyme Q₁₀ by recombinant Escherichia coli, E. coli DH5α (pNTL1).

[0060]FIG. 4 shows HPLC analysis charts showing production of coenzyme Q₁₀ by recombinant Escherichia coli, E. coli JM109 (pUCA1).

BEST MODES FOR CARRYING OUT THE INVENTION EXAMPLE 1

[0061] The chromosomal DNAs of Leucosporidium scotti IFO 1212 and Aspergillus clavatus JCM 1718 were prepared by the method of C. S. Hoffman et al. (Gene, 57 (1987), 267-272). Based on the homology with the known long-chain prenyl diphosphate synthase gene, primers for use in PCR, namely DPS-1 (SEQ ID NO:5 in the sequence listing) and DPS-1 1AS (SEQ ID NO:6 in the sequence listing), were designed. Using these, a PCR cycle of 94° C., 1 minute→43° C., 2 minutes→72° C., 2 minutes, were repeated 40 times after 3 minutes of heat treatment at 94° C., followed by 1.2% agarose gel electrophoresis.

[0062] The thus-obtained fragment, about 220 bp in size, was purified by excising the corresponding gel portion from the gel and then treating with a DNA extraction kit (Sephaglas (trademark) BrandPrep Kit, product of Amersham Pharmacia Biotech), and the purified fragment was cloned into a vector for expression in Escherichia coli using a PCR product direct cloning kit (pT7 BlueT-Vector Kit, product of NOVAGEN) to give pT7-L1DPS and pT7-A1DPS. The DNA base sequence was determined by carrying out the reaction on a DNA sequencer (model 377, product of PerkinElmer) using a DNA sequencing kit (product of PerkinElmer, ABI PRISM (trademark) BigDye (trademark) Terminator Cycle Sequence Ready Reaction Kit with AmptiTaq (registered trademark) DNA polymerase, FS) and according to the manual attached thereto. As a result, a DNA fragment having a base sequence covering the 709th to 915th bases of the base sequence shown under SEQ ID NO:1 in the sequence listing was obtained from Leucosporidium scotti IFO 1212, and a DNA fragment having a base sequence covering the 616th to 822nd bases of the base sequence shown under SEQ ID NO:2 in the sequence listing was obtained from Aspergillus clavatus JCM 1718, respectively. These DNA fragments were estimated to be part of the decaprenyl diphosphate synthase gene since the amino acid sequence “GDFLLXRA” (X is A or G), which is a characteristic region of the long-chain prenyl chain-containing prenyl diphosphate synthase, could be found in the sequence translated from the base sequence determined in the above manner.

EXAMPLE 2

[0063]Leucosporidium scotti IFO 1212 and Aspergillus clavatus JCM 1718 were cultured in 50 mL of 703 medium (5 g/L pepton, 3 g/L yeast extract, 3 g/L malt extract, and 1 g/L glucose, pH 6.0) for 48 hours at 25° C. Then, the cells were harvested by centrifugation at 3,000 revolutions for 20 minutes and frozen immediately in liquid nitrogen.

[0064] The frozen cells were placed in a mortar chilled previously at −70° C. and ground to a powder with a pestle while being kept frozen by occasionally adding liquid nitrogen. Total RNA was prepared from the well-powdered cells using an RNeasy Maxi RNA Purification Kit (product of Qiagen K.K.). Total RNA thus extracted was further purified using an RNeasy Mini RNA Purification Kit (product of Qiagen K.K.). From the purified total RNA, mRNA was prepared using an mRNA purification kit (Ologotex-dT30 <Super> (trademark) mRNA Purification kit, product of Takara Shuzo Co., Ltd.).

EXAMPLE 3

[0065] The DNA fragment containing the region from the DNA obtained in Example 1 to the 3′-terminal region of the decaprenyl diphosphate synthase gene of Leucosporidium scotti IFO 1212 was obtained. RT-PCR was carried out using an RT-PCR kit (High fidelity RNA PCR Kit, product of Takara Shuzo Co., Ltd.) and primer L1S (SEQ ID NO:7 in the sequence listing), which was generated based on the internal sequence of the DNA fragment obtained in Example 1. The thus-obtained fragment, about 850 bp in size, was purified by excising the corresponding gel portion from the gel and then treating with a DNA extraction kit (Sephaglas (trademark) BrandPrep Kit, product of Amersham Pharmacia Biotech), and the purified fragment was cloned into a vector for expression in Escherichia coli using a PCR product direct cloning kit (pT7 BlueT-Vector Kit, product of NOVAGEN) to give pT7-L2DPS. The DNA base sequence was determined by carrying out the reaction on a DNA sequencer (model 377, product of PerkinElmer) using a DNA sequencing kit (product of PerkinElmer, ABI PRISM (trademark) BigDye (trademark) Terminator Cycle Sequence Ready Reaction Kit With AmptiTaq (registered trademark) DNA polymerase, FS) and according to the manual attached thereto.

EXAMPLE 4

[0066] The DNA fragment containing the region from the DNA obtained in Example 1 to the 5′-terminal region of the decaprenyl diphosphate synthase gene of Leucosporidium scotti IFO 1212 was obtained. Reverse transcription was carried out using a 5′-Full RACE Core Set (product of Takara Shuzo Co., Ltd.), the mRNA prepared in Example 2 as template, and primer L7ASP (SEQ ID NO:8 in the sequence listing, “5′-terminal region-phosphorylated”), which was generated based on the internal sequence of the DNA fragment obtained in Example 3, to synthesize a cDNA encoding the 5′-terminal region of the gene containing a part of the fragment obtained in Example 3. Then, the cDNA was circularized using the above kit.

[0067] PCR was carried out using this circularized cDNA as template, and primers L5S (SEQ ID NO:9 in the sequence listing) and L4AS (SEQ ID NO:10 in the sequence listing), which were generated based on the known part of the sequence obtained in Example 3. Furthermore, PCR was carried out to the PCR product using primers L6S (SEQ ID NO:11 in the sequence listing) and L3AS (SEQ ID NO:12 in the sequence listing) to obtain a fragment, about 950 bp in size. The thus-obtained fragment, about 950 bp in size, was purified by excising the corresponding gel portion from the gel and then treating with a DNA extraction kit (Sephaglas (trademark) BrandPrep Kit, product of Amersham Pharmacia Biotech), and the purified fragment was cloned into a vector for expression in Escherichia coli using a PCR product direct cloning kit (pT7 BlueT-Vector Kit, product of NOVAGEN) to give pT7-L3DPS. The DNA base sequence was determined by carrying out the reaction on a DNA sequencer (model 377, product of PerkinElmer) using a DNA sequencing kit (product of PerkinElmer, ABI PRISM (trademark) BigDye (trademark) Terminator Cycle Sequence Ready Reaction Kit With AmptiTaq (registered trademark) DNA polymerase, FS) and according to the manual attached thereto.

EXAMPLE 5

[0068] To obtain the full-length sequence of the decaprenyl diphosphate synthase gene of Leucosporidium scotti IFO 1212, PCR was carried out using pT7-L3DPS as template, primer LN1-2 (SEQ ID NO:13 in the sequence listing), which was generated based on the 5′-terminal sequence of the gene and primer L3AS (SEQ ID NO:12 in the sequence listing) described above to obtain the 5′-terminal region, about 700 bp in size. And, PCR was carried out using pT7-L2DPS as template, primer L1S (SEQ ID NO:7 in the sequence listing) described above and primer LCH (SEQ ID NO:14 in the sequence listing), which was generated based on the 3′-terminal sequence of the gene, to obtain the 3′-terminal region, about 770 bp in size. The two fragments were mixed and denatured, and then slowly cooled to allow annealing, followed by duplex synthesis using DNA polymerase. Subsequently, PCR was carried out using the double-stranded DNA as template, and primers LN1-2 and LCH to obtain DNA containing the full-length sequence of the gene. The base sequence was determined using a DNA sequencer (model 377, product of PerkinElmer, Inc.) and a DNA sequencing kit (product of PerkinElmer, Inc., ABI PRISM (trademark) BigDye (trademark) Terminator Cycle Sequence Ready Reaction Kit With AmptiTaq (registered trademark) DNA polymerase, FS) and according to the manual attached thereto. Thus, the total sequence of the decaprenyl diphosphate synthase gene of Leucosporidium scotti IFO 1212 was revealed. Base sequence was determined on the DNA, about 1.5 kbp in size, and the result is shown under SEQ ID NO:1 in the sequence listing. The amino acid sequence deduced from this base sequence is shown under SEQ ID NO:3.

EXAMPLE 6

[0069] The DNA obtained in Example 5 was cleaved with the restriction enzymes NdeI and HindIII, and the cleavage product was inserted into a vector for expression, pUCNT (described in WO 94/03613), to give a decaprenyl diphosphate synthase gene expression vector, pNTL1. The restriction enzyme map of the thus-obtained expression vector pNTL1 is shown in FIG. 1. The symbol DPS stands for the coding region of the decaprenyl diphosphate synthase gene.

EXAMPLE 7

[0070] The DNA fragment containing the region from the DNA fragment obtained in Example 1 to the 3′-terminal region of the decaprenyl diphosphate synthase gene of Aspergillus clavatus JCM 1718 was obtained. RT-PCR was carried out using an RT-PCR kit (High fidelity RNA PCR Kit, product of Takara Shuzo Co., Ltd.) and primer A1S (SEQ ID NO:15 in the sequence listing), which was generated based on the internal sequence of the DNA fragment obtained in Example 1. The thus-obtained fragment, about 850 bp in size, was purified by excising the corresponding gel portion from the gel and then treating with a DNA extraction kit (Sephaglas (trademark) BrandPrep Kit, product of Amersham Pharmacia Biotech), and the purified fragment was cloned into a vector for expression in Escherichia coli using a PCR product direct cloning kit (pT7 BlueT-Vector Kit, product of NOVAGEN) to give pT7-A2DPS. The DNA base sequence was determined by carrying out the reaction on a DNA sequencer (model 377, product of PerkinElmer) using a DNA sequencing kit (product of PerkinElmer, ABI PRISM (trademark) BigDye (trademark) Terminator Cycle Sequence Ready Reaction Kit With AmptiTaq (registered trademark) DNA polymerase, FS) and according to the manual attached thereto.

EXAMPLE 8

[0071] The DNA fragment containing the region from the DNA fragment obtained in Example 1 to the 5′-terminal region of the decaprenyl diphosphate synthase gene of Aspergillus clavatus JCM 1718 was obtained. Reverse transcription was carried out using a 5′-Full RACE Core Set (product of Takara Shuzo Co., Ltd.), the mRNA prepared in Example 2 as template, and primer A7ASP (SEQ ID NO:16 in the sequence listing, “5′-terminal region-phosphorylated”), which was generated based on the internal sequence of the DNA fragment obtained in Example 7 to synthesize a cDNA encoding the 5′-terminal region of the gene containing a part of the fragment obtained in Example 7. Then, the cDNA was circularized using the above kit.

[0072] PCR was carried out using this circularized cDNA as template, and primers A5S (SEQ ID NO:17 in the sequence listing) and A2AS (SEQ ID NO:18 in the sequence listing), which were generated based on the known part of the sequence obtained in Example 3. Furthermore, PCR was carried out to the PCR product using primers A6S (SEQ ID NO:19 in the sequence listing) and A3AS (SEQ ID NO:20 in the sequence listing) to obtain a fragment, about 850 bp in size. The thus-obtained fragment was purified by excising the corresponding gel portion from the gel and then treating with a DNA extraction kit (Sephaglas (trademark) BrandPrep Kit, product of Amersham Pharmacia Biotech), and the purified fragment was cloned into a vector for expression in Escherichia coli using a PCR product direct cloning kit (pT7 BlueT-Vector Kit, product of NOVAGEN) to give pT7-A3DPS. The DNA base sequence was determined by carrying out the reaction on a DNA sequencer (model 377, product of PerkinElmer) using a DNA sequencing kit (product of PerkinElmer, ABI PRISM (trademark) BigDye (trademark) Terminator Cycle Sequence Ready Reaction Kit With AmptiTaq (registered trademark) DNA polymerase, FS) and according to the manual attached thereto.

EXAMPLE 9

[0073] To obtain the full-length sequence of the decaprenyl diphosphate synthase gene of Aspergillus clavatus JCM 1718, PCR was carried out using pT7-A3DPS as template, primer AN2 (SEQ ID NO:21 in the sequence listing), which was generated based on the 5′-terminal sequence of the gene and primer A3AS (SEQ ID NO:20 in the sequence listing) described above to obtain the 5′-terminal region, about 700 bp in size. And, PCR was carried out using pT7-L2DPS as template, primer A1S (SEQ ID NO:15 in the sequence listing) described above and primer ACH (SEQ ID NO:22 in the sequence listing), which was generated based on the 3′-terminal sequence of the gene, to obtain the 3′-terminal region, about 770 bp in size.

[0074] The two fragments were mixed and denatured, and then slowly cooled to allow annealing, followed by duplex synthesis using DNA polymerase. Subsequently, PCR was carried out using the double-stranded DNA as template, and primers AN2 and ACH to obtain DNA. The base sequence was determined by carrying out the reaction on a DNA sequencer (model 377, product of PerkinElmer, Inc.) using a DNA sequencing kit (product of PerkinElmer, Inc., ABI PRISM (trademark) BigDye (trademark) Terminator Cycle Sequence Ready Reaction Kit With AmptiTaq (registered trademark) DNA polymerase, FS) and according to the manual attached thereto. Thus, the total sequence of the decaprenyl diphosphate synthase gene of Aspergillus clavatus JCM 1718 was revealed. Base sequence was determined on the DNA, about 1.4 kbp in size, and the result is shown under SEQ ID NO:2 in the sequence listing. The amino acid sequence deduced from this DNA sequence is shown under SEQ ID NO:4.

EXAMPLE 10

[0075] The DNA obtained in Example 9 was cleaved with the restriction enzymes BamHI and HindIII, and the cleavage product was inserted into a vector for expression, pUC18, to give a decaprenyl diphosphate synthase gene expression vector, pUCA1. The restriction enzyme map of the thus-obtained expression vector pUCA1 is shown in FIG. 2. The symbol DPS stands for the coding region of the decaprenyl diphosphate synthase gene.

EXAMPLE 11

[0076] The decaprenyl diphosphate synthase gene expression vector pNTL1 was introduced into Escherichia coli DH5α and pUCA1 was introduced into JM109, respectively, to produce recombinant Escherichia coli, E. coli DH5α (pNTL1) and E. coli JM109 (pUCA1). Each recombinant Escherichia coli was shake-cultured overnight in 10 mL of LB medium at 37° C., and cells were harvested by centrifugation (3,000 revolutions, 20 minutes).

[0077] The cells were suspended in 1 mL of a 3% aqueous solution of sulfuric acid and, after 30 minutes of heat treatment at 120° C., 2 mL of a 14% aqueous solution of sodium hydroxide was added, followed by further 15 minutes of heat treatment at 120° C. To the thus-treated suspension was added 3 mL of hexane-isopropanol (10:2) for effecting extraction. After centrifugation, 1.5 mL of the organic solvent layer was separated, and the solvent was evaporated to dryness under reduced pressure conditions. The residue was dissolved in 200 μL of ethanol, and 20 μL of the solution was analyzed by high-performance liquid chromatography (using LC-10A, product of Shimadzu Corp.). For separation, a reversed phase column (YMC-pack ODS-A, 250×4.6 mm, S-5 μm, 120 A) was used, and the coenzyme Q₁₀ formed was detected based on the absorbance at the wavelength 275 nm. The result of E. coli DH5α (pNTL1) (analyzed with mobile phase: methanol/hexane=85/15) is shown in FIG. 3, and the result of E. coli JM109 (pUCA1) (analyzed with mobile phase: ethanol /methanol=2/1) is shown in FIG. 4. As shown in FIGS. 3 and 4, it was revealed that, upon introduction of the decaprenyl diphosphate synthase gene for expression thereof, coenzyme Q₁₀, which is originally not produced in Escherichia coli, could now be produced in recombinant Escherichia coli.

[0078] The thus-obtained recombinant Escherichia coli DH5α (pNTL1) (deposition/accession No. FERM BP-7353) and E. coli JM109(pUCA1) (deposition/accession No. FERM BP-7352) have been deposited, under the Budapest Treaty, with the National Institute of Advanced Industrial Science and Technology International Patent Organism Depositary (zip code: 305-8566 Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, JAPAN) as of Nov. 9, 2000.

INDUSTRIAL APPLICABILITY

[0079] A gene encoding decaprenyl diphosphate synthase, which is the key enzyme in the biosynthesis of coenzyme Q₁₀, was isolated from a fungal species belonging to the genus Leucosporidium and genus Aspergillus and it was sequenced. This could successfully be introduced in Escherichia coli for expression thereof. Furthermore, improvements in gene sequence successfully resulted in-production in significant amounts. By using the method of the invention, it becomes possible to efficiently produce coenzyme Q₁₀, which is in use as a drug, among others.

1 22 1 1506 DNA Leucosporidium scottii 1 atg tcg cgg aca ctg ccg ata tct cgc ttg aga gga cgt gca cgg cca 48 Met Ser Arg Thr Leu Pro Ile Ser Arg Leu Arg Gly Arg Ala Arg Pro 1 5 10 15 tct tcg agt cta cta cag ctc cca act gag ctg caa aag ctc tcc tcc 96 Ser Ser Ser Leu Leu Gln Leu Pro Thr Glu Leu Gln Lys Leu Ser Ser 20 25 30 tcc cca acc tca tcc ctc cgt cat gct tcc cct tcc cgc tcc gcc tgg 144 Ser Pro Thr Ser Ser Leu Arg His Ala Ser Pro Ser Arg Ser Ala Trp 35 40 45 act tca gcc atc ccc ggt ctc tcg tct gcc acc cca ttc gct tcg act 192 Thr Ser Ala Ile Pro Gly Leu Ser Ser Ala Thr Pro Phe Ala Ser Thr 50 55 60 tca acc tct tcc tcc ctc ctc gct ggc tca tcc aaa gta gcg ttg caa 240 Ser Thr Ser Ser Ser Leu Leu Ala Gly Ser Ser Lys Val Ala Leu Gln 65 70 75 80 gat ccc ctc aag ccg cta ggc gca gag atg ggc ttg ctg agg tcc aac 288 Asp Pro Leu Lys Pro Leu Gly Ala Glu Met Gly Leu Leu Arg Ser Asn 85 90 95 gtc cag cac ctc ctt ggt tca ggt cat cca gca ctg gat acc atc gcc 336 Val Gln His Leu Leu Gly Ser Gly His Pro Ala Leu Asp Thr Ile Ala 100 105 110 aag tac tac ttt caa gcc gaa ggg aag cat gtt cga ccg atg ctc atc 384 Lys Tyr Tyr Phe Gln Ala Glu Gly Lys His Val Arg Pro Met Leu Ile 115 120 125 ttg ctc atg agc caa gcg acg aat gga ctc gca ccc ggc tgg gaa cag 432 Leu Leu Met Ser Gln Ala Thr Asn Gly Leu Ala Pro Gly Trp Glu Gln 130 135 140 agg cgg gat caa gcg gca gca gcg gaa ctg aag agg gag caa ggc gac 480 Arg Arg Asp Gln Ala Ala Ala Ala Glu Leu Lys Arg Glu Gln Gly Asp 145 150 155 160 gaa gga tta gga ggg gac gat atc gac gaa cct cta agc cca cct tcc 528 Glu Gly Leu Gly Gly Asp Asp Ile Asp Glu Pro Leu Ser Pro Pro Ser 165 170 175 gtc ctc aac gac caa aac ccc tcg atg ctc gct tcg gcc aaa tcg ttc 576 Val Leu Asn Asp Gln Asn Pro Ser Met Leu Ala Ser Ala Lys Ser Phe 180 185 190 ttc tcc gac cct ctc gct tcg ctc cga ccc gct ccc act ccc act tcc 624 Phe Ser Asp Pro Leu Ala Ser Leu Arg Pro Ala Pro Thr Pro Thr Ser 195 200 205 atc gcc caa tca atc cat caa act cac ctc ctt ccc tcc caa cgt cgt 672 Ile Ala Gln Ser Ile His Gln Thr His Leu Leu Pro Ser Gln Arg Arg 210 215 220 ctc gcc gaa atc acc gaa atg att cac gtc gcc tcg ttg ctg cac gac 720 Leu Ala Glu Ile Thr Glu Met Ile His Val Ala Ser Leu Leu His Asp 225 230 235 240 gat gtt att gac ctc gca gag acg agg cga tcg gcc ccc tca gct cct 768 Asp Val Ile Asp Leu Ala Glu Thr Arg Arg Ser Ala Pro Ser Ala Pro 245 250 255 tcg ctc ttt ggc aac aag ctc tcc atc ctc gcg gga gat ttc ttg ctc 816 Ser Leu Phe Gly Asn Lys Leu Ser Ile Leu Ala Gly Asp Phe Leu Leu 260 265 270 gcc cga gct tcc ctc gct ctc tcg agg ttg ggg agc aat gag gta gtc 864 Ala Arg Ala Ser Leu Ala Leu Ser Arg Leu Gly Ser Asn Glu Val Val 275 280 285 gag ctc gtc gct tcc gtt ctc gcc aac ttg gtc gag ggg gag gtt atg 912 Glu Leu Val Ala Ser Val Leu Ala Asn Leu Val Glu Gly Glu Val Met 290 295 300 cag atg aag ggg aac gta ccg ggc aag gaa ggg ctg ttg gca ggg gca 960 Gln Met Lys Gly Asn Val Pro Gly Lys Glu Gly Leu Leu Ala Gly Ala 305 310 315 320 gga gga gga tca aca gcc aag gga ccg aca ccc gag atc ttc gac cac 1008 Gly Gly Gly Ser Thr Ala Lys Gly Pro Thr Pro Glu Ile Phe Asp His 325 330 335 tac atg aag aag acg tac ctc aag acg gcg agc ctg att gcc aaa agt 1056 Tyr Met Lys Lys Thr Tyr Leu Lys Thr Ala Ser Leu Ile Ala Lys Ser 340 345 350 acg agg gcg acg acg att cta ggt gga tgt gga gtc aag cag gga tgg 1104 Thr Arg Ala Thr Thr Ile Leu Gly Gly Cys Gly Val Lys Gln Gly Trp 355 360 365 gca gag gga gag aag gtc aag gat atc gcc tac tcg tat ggt cgt aac 1152 Ala Glu Gly Glu Lys Val Lys Asp Ile Ala Tyr Ser Tyr Gly Arg Asn 370 375 380 ttg ggc atc gcc ttc cag ctc gtg gac gac atg ctc gac ttt acg gca 1200 Leu Gly Ile Ala Phe Gln Leu Val Asp Asp Met Leu Asp Phe Thr Ala 385 390 395 400 tca gca gca caa ctc ggc aaa cca gga gga gga gcc gac ctc aaa ctc 1248 Ser Ala Ala Gln Leu Gly Lys Pro Gly Gly Gly Ala Asp Leu Lys Leu 405 410 415 ggt ctc gct acc gca cca gca ctc tac gcg tgg gag gaa ttc ccc gaa 1296 Gly Leu Ala Thr Ala Pro Ala Leu Tyr Ala Trp Glu Glu Phe Pro Glu 420 425 430 ttg ggg gcg atg att gag cgc aag ttt gct ggc gag gac gat gtc gag 1344 Leu Gly Ala Met Ile Glu Arg Lys Phe Ala Gly Glu Asp Asp Val Glu 435 440 445 cag gcc cga cac ctc atc tcg cgc tcc tcc ggg gcc gaa cga acg gcc 1392 Gln Ala Arg His Leu Ile Ser Arg Ser Ser Gly Ala Glu Arg Thr Ala 450 455 460 gct ctc gcc gcc gag cac tca aaa ttg gcg cgt caa gcg ctc gaa ggt 1440 Ala Leu Ala Ala Glu His Ser Lys Leu Ala Arg Gln Ala Leu Glu Gly 465 470 475 480 ctc ccc gat agc gag gcg agg aca gca ttg gat aac atg gcg agg gat 1488 Leu Pro Asp Ser Glu Ala Arg Thr Ala Leu Asp Asn Met Ala Arg Asp 485 490 495 aca ttg tcg agg aag aag 1506 Thr Leu Ser Arg Lys Lys 500 2 1353 DNA Aspergillus clavatus 2 atg aga gct cga acg gtc tcg gcc tct ggc ctc att ctc tca tcg cga 48 Met Arg Ala Arg Thr Val Ser Ala Ser Gly Leu Ile Leu Ser Ser Arg 1 5 10 15 acg acg acc tcg acc tcg ata tgc tgg caa tgc ctt cgt gaa gat ctc 96 Thr Thr Thr Ser Thr Ser Ile Cys Trp Gln Cys Leu Arg Glu Asp Leu 20 25 30 ctc tca aat caa gtt caa atc cac gtt cga aaa tac cat ccc acc cgc 144 Leu Ser Asn Gln Val Gln Ile His Val Arg Lys Tyr His Pro Thr Arg 35 40 45 cgg aaa gat gtc tct ccc ttc ggt gcc gcc gtt tct gca gcg cag acc 192 Arg Lys Asp Val Ser Pro Phe Gly Ala Ala Val Ser Ala Ala Gln Thr 50 55 60 atc ttc aaa ggc ctg cca aag gct cct ccg ggg atc tcg gta gat cca 240 Ile Phe Lys Gly Leu Pro Lys Ala Pro Pro Gly Ile Ser Val Asp Pro 65 70 75 80 ttg agg atc gtg ggg aaa gag ctc aag ttt ttg acg aag aat ata cgc 288 Leu Arg Ile Val Gly Lys Glu Leu Lys Phe Leu Thr Lys Asn Ile Arg 85 90 95 caa ttg ctg ggt tcg ggc cac ccg act ctt gat aaa gtg gcc aaa tat 336 Gln Leu Leu Gly Ser Gly His Pro Thr Leu Asp Lys Val Ala Lys Tyr 100 105 110 tac acc cgc agc gag ggc aaa cat atg cgt ccg ctt ttg gtc ctg ctc 384 Tyr Thr Arg Ser Glu Gly Lys His Met Arg Pro Leu Leu Val Leu Leu 115 120 125 atg tca cag gcg acg gcg ttg act ccg cgg cag agt cgt tca aac ttc 432 Met Ser Gln Ala Thr Ala Leu Thr Pro Arg Gln Ser Arg Ser Asn Phe 130 135 140 acc cct tca cag atg gtc aat gat ccc att agc tcg cct tcc gtc ctc 480 Thr Pro Ser Gln Met Val Asn Asp Pro Ile Ser Ser Pro Ser Val Leu 145 150 155 160 gcc gat acg aac ccg gat ctc agc ccg ctt gtc tcg aaa tcg gcc gaa 528 Ala Asp Thr Asn Pro Asp Leu Ser Pro Leu Val Ser Lys Ser Ala Glu 165 170 175 gcg caa tat gat ttt gcg ggg gat gag aat acc ctg cct acg cag cgc 576 Ala Gln Tyr Asp Phe Ala Gly Asp Glu Asn Thr Leu Pro Thr Gln Arg 180 185 190 cga ctc gct gag atc acg gaa ttg atc cat acc gcc tcg ctc ctc cac 624 Arg Leu Ala Glu Ile Thr Glu Leu Ile His Thr Ala Ser Leu Leu His 195 200 205 gac gac gtg atc gac aac gct gtt act cgg agg tcg tct aac tcc gca 672 Asp Asp Val Ile Asp Asn Ala Val Thr Arg Arg Ser Ser Asn Ser Ala 210 215 220 aac ctc cag ttt gga aat aag atg gcc gtc ctg gcc gga gat ttc ctg 720 Asn Leu Gln Phe Gly Asn Lys Met Ala Val Leu Ala Gly Asp Phe Leu 225 230 235 240 ctc ggc cga gct tcc gtc gcc ctg gcg cgc ctg aga gac ccc gag gtc 768 Leu Gly Arg Ala Ser Val Ala Leu Ala Arg Leu Arg Asp Pro Glu Val 245 250 255 aca gaa ctg ctt gca act gtc att gcc aac ctg gtg gag gga gag ttc 816 Thr Glu Leu Leu Ala Thr Val Ile Ala Asn Leu Val Glu Gly Glu Phe 260 265 270 atg caa ttg aag aat acc gcc gcg gat gag aag aac ccc gtg ttc acc 864 Met Gln Leu Lys Asn Thr Ala Ala Asp Glu Lys Asn Pro Val Phe Thr 275 280 285 gac ggg acc atc tcg tac tac ttg caa aag acg tac ctc aag acc gcc 912 Asp Gly Thr Ile Ser Tyr Tyr Leu Gln Lys Thr Tyr Leu Lys Thr Ala 290 295 300 agt ctg atc agc aag tcg tgc cgt gca gcg gca ttg cta ggt ggc agt 960 Ser Leu Ile Ser Lys Ser Cys Arg Ala Ala Ala Leu Leu Gly Gly Ser 305 310 315 320 acg cct gag gtt gtc gat gct gct tat gcc tat gga cgc aac ctg ggc 1008 Thr Pro Glu Val Val Asp Ala Ala Tyr Ala Tyr Gly Arg Asn Leu Gly 325 330 335 ctg gca ttc cag ctg gtg gat gat ctg ctg gat tac acc gtg agt ggg 1056 Leu Ala Phe Gln Leu Val Asp Asp Leu Leu Asp Tyr Thr Val Ser Gly 340 345 350 gtt gag tta ggc aag cct gcc gga gcc gat ctg gag ctg ggt ctt gcg 1104 Val Glu Leu Gly Lys Pro Ala Gly Ala Asp Leu Glu Leu Gly Leu Ala 355 360 365 act gct ccg ctg ctc ttt gcc tgg aag cag aac cct gag ctg ggc ccc 1152 Thr Ala Pro Leu Leu Phe Ala Trp Lys Gln Asn Pro Glu Leu Gly Pro 370 375 380 ttg gtc ggt cgg aag ttc agc cga gag ggg gat gta caa atg gct cgt 1200 Leu Val Gly Arg Lys Phe Ser Arg Glu Gly Asp Val Gln Met Ala Arg 385 390 395 400 gaa ctg gtg tac aag agc gat ggc gtt gaa cag acc cgc gct ctg gcc 1248 Glu Leu Val Tyr Lys Ser Asp Gly Val Glu Gln Thr Arg Ala Leu Ala 405 410 415 cag gag tac gcc gac aag gcc att acc gcc gtc agc aac ttc cct gac 1296 Gln Glu Tyr Ala Asp Lys Ala Ile Thr Ala Val Ser Asn Phe Pro Asp 420 425 430 agt gaa gcc aag gct ggt ctc atc caa atg tgc gag aaa gcc atg aac 1344 Ser Glu Ala Lys Ala Gly Leu Ile Gln Met Cys Glu Lys Ala Met Asn 435 440 445 cgg aga aag 1353 Arg Arg Lys 450 3 502 PRT Leucosporidium scottii 3 Met Ser Arg Thr Leu Pro Ile Ser Arg Leu Arg Gly Arg Ala Arg Pro 1 5 10 15 Ser Ser Ser Leu Leu Gln Leu Pro Thr Glu Leu Gln Lys Leu Ser Ser 20 25 30 Ser Pro Thr Ser Ser Leu Arg His Ala Ser Pro Ser Arg Ser Ala Trp 35 40 45 Thr Ser Ala Ile Pro Gly Leu Ser Ser Ala Thr Pro Phe Ala Ser Thr 50 55 60 Ser Thr Ser Ser Ser Leu Leu Ala Gly Ser Ser Lys Val Ala Leu Gln 65 70 75 80 Asp Pro Leu Lys Pro Leu Gly Ala Glu Met Gly Leu Leu Arg Ser Asn 85 90 95 Val Gln His Leu Leu Gly Ser Gly His Pro Ala Leu Asp Thr Ile Ala 100 105 110 Lys Tyr Tyr Phe Gln Ala Glu Gly Lys His Val Arg Pro Met Leu Ile 115 120 125 Leu Leu Met Ser Gln Ala Thr Asn Gly Leu Ala Pro Gly Trp Glu Gln 130 135 140 Arg Arg Asp Gln Ala Ala Ala Ala Glu Leu Lys Arg Glu Gln Gly Asp 145 150 155 160 Glu Gly Leu Gly Gly Asp Asp Ile Asp Glu Pro Leu Ser Pro Pro Ser 165 170 175 Val Leu Asn Asp Gln Asn Pro Ser Met Leu Ala Ser Ala Lys Ser Phe 180 185 190 Phe Ser Asp Pro Leu Ala Ser Leu Arg Pro Ala Pro Thr Pro Thr Ser 195 200 205 Ile Ala Gln Ser Ile His Gln Thr His Leu Leu Pro Ser Gln Arg Arg 210 215 220 Leu Ala Glu Ile Thr Glu Met Ile His Val Ala Ser Leu Leu His Asp 225 230 235 240 Asp Val Ile Asp Leu Ala Glu Thr Arg Arg Ser Ala Pro Ser Ala Pro 245 250 255 Ser Leu Phe Gly Asn Lys Leu Ser Ile Leu Ala Gly Asp Phe Leu Leu 260 265 270 Ala Arg Ala Ser Leu Ala Leu Ser Arg Leu Gly Ser Asn Glu Val Val 275 280 285 Glu Leu Val Ala Ser Val Leu Ala Asn Leu Val Glu Gly Glu Val Met 290 295 300 Gln Met Lys Gly Asn Val Pro Gly Lys Glu Gly Leu Leu Ala Gly Ala 305 310 315 320 Gly Gly Gly Ser Thr Ala Lys Gly Pro Thr Pro Glu Ile Phe Asp His 325 330 335 Tyr Met Lys Lys Thr Tyr Leu Lys Thr Ala Ser Leu Ile Ala Lys Ser 340 345 350 Thr Arg Ala Thr Thr Ile Leu Gly Gly Cys Gly Val Lys Gln Gly Trp 355 360 365 Ala Glu Gly Glu Lys Val Lys Asp Ile Ala Tyr Ser Tyr Gly Arg Asn 370 375 380 Leu Gly Ile Ala Phe Gln Leu Val Asp Asp Met Leu Asp Phe Thr Ala 385 390 395 400 Ser Ala Ala Gln Leu Gly Lys Pro Gly Gly Gly Ala Asp Leu Lys Leu 405 410 415 Gly Leu Ala Thr Ala Pro Ala Leu Tyr Ala Trp Glu Glu Phe Pro Glu 420 425 430 Leu Gly Ala Met Ile Glu Arg Lys Phe Ala Gly Glu Asp Asp Val Glu 435 440 445 Gln Ala Arg His Leu Ile Ser Arg Ser Ser Gly Ala Glu Arg Thr Ala 450 455 460 Ala Leu Ala Ala Glu His Ser Lys Leu Ala Arg Gln Ala Leu Glu Gly 465 470 475 480 Leu Pro Asp Ser Glu Ala Arg Thr Ala Leu Asp Asn Met Ala Arg Asp 485 490 495 Thr Leu Ser Arg Lys Lys 500 4 451 PRT Aspergillus clavatus 4 Met Arg Ala Arg Thr Val Ser Ala Ser Gly Leu Ile Leu Ser Ser Arg 1 5 10 15 Thr Thr Thr Ser Thr Ser Ile Cys Trp Gln Cys Leu Arg Glu Asp Leu 20 25 30 Leu Ser Asn Gln Val Gln Ile His Val Arg Lys Tyr His Pro Thr Arg 35 40 45 Arg Lys Asp Val Ser Pro Phe Gly Ala Ala Val Ser Ala Ala Gln Thr 50 55 60 Ile Phe Lys Gly Leu Pro Lys Ala Pro Pro Gly Ile Ser Val Asp Pro 65 70 75 80 Leu Arg Ile Val Gly Lys Glu Leu Lys Phe Leu Thr Lys Asn Ile Arg 85 90 95 Gln Leu Leu Gly Ser Gly His Pro Thr Leu Asp Lys Val Ala Lys Tyr 100 105 110 Tyr Thr Arg Ser Glu Gly Lys His Met Arg Pro Leu Leu Val Leu Leu 115 120 125 Met Ser Gln Ala Thr Ala Leu Thr Pro Arg Gln Ser Arg Ser Asn Phe 130 135 140 Thr Pro Ser Gln Met Val Asn Asp Pro Ile Ser Ser Pro Ser Val Leu 145 150 155 160 Ala Asp Thr Asn Pro Asp Leu Ser Pro Leu Val Ser Lys Ser Ala Glu 165 170 175 Ala Gln Tyr Asp Phe Ala Gly Asp Glu Asn Thr Leu Pro Thr Gln Arg 180 185 190 Arg Leu Ala Glu Ile Thr Glu Leu Ile His Thr Ala Ser Leu Leu His 195 200 205 Asp Asp Val Ile Asp Asn Ala Val Thr Arg Arg Ser Ser Asn Ser Ala 210 215 220 Asn Leu Gln Phe Gly Asn Lys Met Ala Val Leu Ala Gly Asp Phe Leu 225 230 235 240 Leu Gly Arg Ala Ser Val Ala Leu Ala Arg Leu Arg Asp Pro Glu Val 245 250 255 Thr Glu Leu Leu Ala Thr Val Ile Ala Asn Leu Val Glu Gly Glu Phe 260 265 270 Met Gln Leu Lys Asn Thr Ala Ala Asp Glu Lys Asn Pro Val Phe Thr 275 280 285 Asp Gly Thr Ile Ser Tyr Tyr Leu Gln Lys Thr Tyr Leu Lys Thr Ala 290 295 300 Ser Leu Ile Ser Lys Ser Cys Arg Ala Ala Ala Leu Leu Gly Gly Ser 305 310 315 320 Thr Pro Glu Val Val Asp Ala Ala Tyr Ala Tyr Gly Arg Asn Leu Gly 325 330 335 Leu Ala Phe Gln Leu Val Asp Asp Leu Leu Asp Tyr Thr Val Ser Gly 340 345 350 Val Glu Leu Gly Lys Pro Ala Gly Ala Asp Leu Glu Leu Gly Leu Ala 355 360 365 Thr Ala Pro Leu Leu Phe Ala Trp Lys Gln Asn Pro Glu Leu Gly Pro 370 375 380 Leu Val Gly Arg Lys Phe Ser Arg Glu Gly Asp Val Gln Met Ala Arg 385 390 395 400 Glu Leu Val Tyr Lys Ser Asp Gly Val Glu Gln Thr Arg Ala Leu Ala 405 410 415 Gln Glu Tyr Ala Asp Lys Ala Ile Thr Ala Val Ser Asn Phe Pro Asp 420 425 430 Ser Glu Ala Lys Ala Gly Leu Ile Gln Met Cys Glu Lys Ala Met Asn 435 440 445 Arg Arg Lys 450 5 24 DNA Artificial Sequence Description of Artificial Sequence primer DPS-1 5 aaggatcctn ytncaygayg aygt 24 6 17 DNA Artificial Sequence Description of Artificial Sequence primer DPS-1 1AS 6 arytgnadra aytcncc 17 7 21 DNA Artificial Sequence Description of Artificial Sequence primer L1S 7 gcagagacga ggcgatcggc c 21 8 15 DNA Artificial Sequence Description of Artificial Sequence primer L7ASP 8 ggcctgctcg acatc 15 9 21 DNA Artificial Sequence Description of Artificial Sequence primer L5S 9 tgggcagagg gagagaaggt c 21 10 21 DNA Artificial Sequence Description of Artificial Sequence primer L4AS 10 gttggcgaga acggaagcga c 21 11 21 DNA Artificial Sequence Description of Artificial Sequence primer L6S 11 gcaccagcac tctacgcgtg g 21 12 21 DNA Artificial Sequence Description of Artificial Sequence primer L3AS 12 gttgccaaag agcgaaggag c 21 13 33 DNA Artificial Sequence Description of Artificial Sequence primer LN1-2 13 ttgcttctct cgcatatgtc gcggacactg ccg 33 14 29 DNA Artificial Sequence Description of Artificial Sequence primer LCH 14 acaagcttct acttcttcct cgacaatgt 29 15 21 DNA Artificial Sequence Description of Artificial Sequence primer A1S 15 gttactcgga ggtcgtctaa c 21 16 15 DNA Artificial Sequence Description of Artificial Sequence primer A7ASP 16 caccagttca cgagc 15 17 21 DNA Artificial Sequence Description of Artificial Sequence primer A5S 17 gttgtcgatg ctgcttatgc c 21 18 21 DNA Artificial Sequence Description of Artificial Sequence primer A2AS 18 aatgacagtt gcaagcagtt c 21 19 21 DNA Artificial Sequence Description of Artificial Sequence primer A6S 19 gcctggaagc agaaccctga g 21 20 21 DNA Artificial Sequence Description of Artificial Sequence primer A3AS 20 caggacggcc atcttatttc c 21 21 33 DNA Artificial Sequence Description of Artificial Sequence primer AN2 21 aaggatccga tgagagctcg aacggtctcg gcc 33 22 29 DNA Artificial Sequence Description of Artificial Sequence primer ACH 22 acaagcttct actttctccg gttcatggc 29 

1. A DNA of the following (a), (b) or (c): (a) a DNA whose base sequence is as described under SEQ ID NO:1; (b) a DNA having a base sequence derived from the base sequence shown under SEQ ID NO:1 by deletion, addition, insertion and/or substitution of one or several bases and encoding a protein having decaprenyl diphosphate synthase activity; (c) a DNA capable of hybridizing with a DNA comprising the base sequence shown under SEQ ID NO:1 under stringent conditions and encoding a protein having decaprenyl diphosphate synthase activity:
 2. A DNA of the following (d), (e) or (f): (d) a DNA whose base sequence is as described under SEQ ID NO:2; (e) a DNA having a base sequence derived from the base sequence shown under SEQ ID NO:2 by deletion, addition, insertion and/or substitution of one or several bases and encoding a protein having decaprenyl diphosphate synthase activity; (f) a DNA capable of hybridizing with a DNA comprising the base sequence shown under SEQ ID NO:2 under stringent conditions and encoding a protein having decaprenyl diphosphate synthase activity.
 3. A protein of the following (g) or (h): (g) a protein whose amino acid sequence is as described under SEQ ID NO:3; (h) a protein having an amino acid sequence derived from the amino acid sequence shown under SEQ ID NO:3 by deletion, addition, insertion and/or substitution of one or several amino acid residues and having decaprenyl diphosphate synthase activity.
 4. A protein of the following (i) or (j): (i) a protein whose amino acid sequence is as described under SEQ ID NO:4; (j) a protein having an amino acid sequence derived from the amino acid sequence shown under SEQ ID NO:4 by deletion, addition, insertion and/or substitution of one or several amino acid residues and having decaprenyl diphosphate synthase activity.
 5. A DNA encoding the protein according to claim
 3. 6. A DNA encoding the protein according to claim
 4. 7. An expression vector resulting from insertion of the DNA according to claim 1 or 5 into a vector for expression.
 8. An expression vector resulting from insertion of the DNA according to claim 2 or 6 into a vector for expression.
 9. The expression vector according to claim 7, wherein the vector for expression is pUCNT.
 10. The expression vector according to claim 8, wherein the vector for expression is pUC18.
 11. The expression vector according to claim 9, which is pNTL1.
 12. The expression vector according to claim 10, which is pUCA1.
 13. A transformant resulting from transformation of a host microorganism with the DNA according to claim 1, 2, 5 or
 6. 14. A transformant resulting from transformation of a host microorganism with the expression vector according to claim 7, 8, 9, 10, 11 or
 12. 15. The transformant according to claim 13 or 14, wherein the host microorganism is a strain of Escherichia coli.
 16. The transformant according to claim 15, wherein the strain of Escherichia coli is Escherichia coli DH5α or JM109.
 17. The transformant according to claim 16, which is E. coli DH5α (pNTL1) (FRPM BP-7353).
 18. The transformant according to claim 16, which is E. coli JM109(pUCA1) (FRPM BP-7352).
 19. A process for producing coenzyme Q₁₀, which comprises cultivating the transformant according to claim 13, 14, 15, 16, 17 or 18 in a medium and recovering coenzyme Q₁₀ thus formed and accumulated in the medium. 